PROJECT SUMMARY Striatal dopamine (DA) transmission is crucial for movement, motivation and reward, but is critically disrupted in neurodegenerative, neuropsychiatric and substance abuse disorders. Despite the importance of DA signaling in both health and disease, its mechanisms remain poorly understood. Striatal DA receptors are often located outside of synapses and striatal DA release sites are not always associated with post-synaptic structures. These observations have led to the belief that DA signaling occurs by ?volume transmission? whereby low concentrations of DA diffuse from release sites and non-selectively activate receptors across a large sphere of influence. However, recent evidence has challenged this concept. Our previous work has demonstrated that DA release drives the rapid activation of D2 receptors and D2 receptor-mediated inhibitory post-synaptic currents (D2-IPSCs) in striatal medium spiny neurons (MSNs). Furthermore, others have shown that striatal DA release only occurs at a small proportion of DA varicosities (20-30%) which requires specialized machinery (active zones) which typically enables highly precise forms of neurotransmission. These findings suggest that striatal DA signaling occurs in a spatially-restricted manner. Consistent with this idea, recent work has demonstrated that DA release occurs from highly localized ?hotspots? detected with genetically-encoded fluorescent DA sensors. Considering the high density of diverse inputs which are integrated in the striatum, the spatial organization DA signaling is critical for striatal output. However, the organization of DA release sites and post-synaptic receptors remains unclear. Furthermore, drugs of abuse (e.g. cocaine) increase the spread of DA signaling by preventing its uptake. Considering this effect, the contribution of uptake to DA signaling requires further elucidation. This study will utilize a novel approach combining high resolution, two-photon microscopy of a genetically- encoded fluorescent DA sensor, with synaptic electrophysiology to: (1) definine the dynamics and organization of DA neurotransmission in the dorsal striatum; and (2) determine the contribution of diffusion and uptake to DA signaling. These experiments will test the hypothesis that that DA transmission occurs at specialized ?synapse-like? structures in a point-to-point manner. Better illumination of DA signaling and its associated microarchitecture will allow better understanding of pathological changes which occur at DA synapses underlying substance abuse and drug addiction.